Therapeutic intervention with nucleic acids is a promising strategy for the treatment of human disease. The safe and effective delivery of nucleic acids into cells, however, remains a significant obstacle. Virus-based delivery has been investigated thoroughly and has shown good efficacy yet concerns about toxicity and immunogenicity limit the clinical potential of this approach. Non-viral vectors such as cationic polymers are now receiving increased attention as effective nucleic acid delivery agents because of their demonstrated delivery potential. In order for these synthetic vectors to be clinically effective, they must meet several criteria: 1) form particles (e.g., nanoparticles) with nucleic acids, 2) mediate delivery into the target cells of interest (transfection) 3) low toxicity, low immunogenicity, and biodegradability. Although much progress has been made, significant challenges remain as to date there are still no FDA approved treatments utilizing RNAi interference (siRNA) or for gene (DNA) therapy. Increasing the chemical diversity of available polymers may assist in the development of new safe and effective materials, and increase the probability of clinical success.
Despite promise in the laboratory, the potential of genetic therapies for the treatment of disease has yet to be realized. Initial attempts to translate genetic materials into cures led to cancer and, in some cases, death to patients involved in the clinical trials. Such deleterious outcomes were attributed not to the genetic material, but to the viral delivery systems utilized in these trials. As a result, there has been intense interest in developing synthetic materials that have the delivery efficiencies of viral vectors but circumvent the mutagenesis that led to the observed side effects (e.g., cancer).
Synthetic materials, or nonviral delivery vectors, come in a variety of forms that work in unique ways. Polymeric materials such as polyethylenimine or poly(beta-amino ester)s have been shown to efficiently complex DNA for delivery into the cell. Polymers in these classes of delivery agents typically contain amine functionalities that serve to electrostatically bind to DNA to form nanoparticles that are then taken up by the cell via endocytosis. Once in the cell, these amine groups serve to buffer the endosome and cause an influx of ions due to the proton-sponge mechanism. The resulting burst of the endocytic vesicle leads to the release of the payload of the particle, which is then free to travel to the nucleus where the DNA is expressed.
While the mechanism of RNA-based therapies is different, the objective of the delivery system remains the same. The RNA must be complexed and internalized by the cell in order to exhibit activity. In many cases, polymeric materials do not work as efficiently for RNA delivery. This is likely due to the difference in chemical structure of the therapeutic RNA being delivered, which are generally short, linear fragments containing additional hydroxyl moieties on each ribose ring. These differences necessitate an alternative nonviral approach that is suited for complexation with short RNA strands. Promising results have been achieved with materials that form liposomes or lipoplexes that entrap the RNA or form nanoparticles, which are efficiently internalized by the cell.
The materials utilized to form a lipid-based delivery system generally consist of a positively charged headgroup and a hydrophobic tail. The charged portion serves to electrostatically bind the negatively charged RNA, while the hydrophobic tail leads to self-assembly into lipophilic particles. Such cationic lipids are promising but still fall short of the transfection efficiency achieved by viral vectors. Few advances have been made in the field, in part due to the limited structural diversity of these lipid or polymeric molecules, which is a result of the difficult synthetic procedures required to access these structures. Therefore, in order to push the area of nonviral particle delivery systems forward, it is necessary to investigate chemical transformations that can lead to diverse molecules capable of complexing RNA and shuttling the material across the cell membrane. The most successful approach to date has been the contribution by Anderson and coworkers, who generated a library of lipid-like cationic materials and polymers using straightforward simple chemical transformations. See, e.g., PCT Application Publication Nos. WO 2004/106411; WO 2006/138380; WO 2007/143659; WO 2008/011561; and WO 2010/053572. The Anderson team generated over 1000 cationic materials that were tested for their ability to complex and deliver RNA in a high throughput assay. This screen led to the identification of a number of lead cationic materials that were more efficient in vitro than the current industry standard, Lipofectamine 2000, and are currently being tested in vivo for potential use in therapeutic applications. See, e.g., Akinc et al., Nat. Biotech. 2008 26:561.